Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing

Research output: Contribution to journalArticle

Abstract

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA trans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determining trans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissive trans-splicing sequences targeting an intron in the Synapsin I gene. Using viral vectors, we were able to splice full-length fluorophores into the mRNA while retaining very low off-target expression. Furthermore, this approach also showed evidence of functionality in the mouse striatum. However, in its current form, the trans-splicing events are stochastic and the overall activity lower than would be required for therapies targeting loss-of-function mutations. Nevertheless, the herein described barcode-based screening assay provides a unique possibility to screen and map large libraries in single animals or cell assays with very high precision.

Details

Authors
Organisations
External organisations
  • Lund University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medical Genetics

Keywords

  • Barcoding, Plasmid library, Trans-splicing, Viral vectors
Original languageEnglish
Pages (from-to)673-687
Number of pages15
JournalRNA
Volume24
Issue number5
Publication statusPublished - 2018 May 1
Publication categoryResearch
Peer-reviewedYes